Hepatic formation of bile plays a critical role in the maintenance of metabolic homeostasis. Despite a substantial research effort in recent years, the mechanisms and pathways by which biliary constituents are transported through hepatocytes are still largely unknown. In the proposed research program, our aims are to probe the intracellular events involved in the formation of hepatic bile. Specifically, we propose to: 1) define the uptake and metabolism of fluorescent lipid and bile salt probes by isolated liver and hepatocyte doublet preparations; 2) to define the intracellular organelles and pathways involved in the delivery of fluorescent lipids and bile salts to the canalicular pole of hepatocytes; and 3) to explore the modulation of intracellular lipid and bile salt fluorophore transport by ambient physiologic and pathophysiologic conditions. Rigorous control studies will include characterization of the purity and in vitro behavior of each lipid and bile salt fluorescent probe, and evaluation of the uptake, metabolism and excretion of liposomal and albumin-bound fluorophores by the isolated perfused liver, hepatocytes and sinusoidal cells, and determination of the relative affinity of subcellular fractions of the liver for each probe. The intracellular membrane and organelle distribution of fluorescent lipids and bile salts will then be examined of isolated rat hepatocyte doublets by fluorescence microscopys. Intracellular distribution patterns will be correlated with cellular structure, using co-localization techniques for subcellular organelles such as the endoplasmic reticulum and Golgi apparatus. The effects of graded doses of bile salts of different hydrophobicity and the effects of cytoskeleton inhibitors, including colchicine and phalloidin, will be examined to determine the role of transhepatic bile salt flux and the cytoskeletal system in facilitating excretion of biliary constituents. These studies should not only provide information critical for the characterization of intracellular mechanisms of bile formation, but also will have relevance for three important areas of human disease: cholestasis or the failure of bile formation, which is a common form of liver dysfunction; the formation of abnormal bile, which may lead to gallstone formation; and the removal of drugs and toxic compounds from the blood for excretion into bile.